JCB: Review

Telomere-driven diseases and telomere-targeting therapies

Paula Martínez and Maria A. Blasco

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre, Madrid E-28029, Spain

Telomeres, the protective ends of linear chromosomes, structure has been proposed to protect chromosome ends from shorten throughout an individual’s lifetime. Telomere degradation and DNA repair activities as well as from telomer- ase activity (Fig. 1, A and B; Griffith et al., 1999; Doksani et al., shortening is proposed to be a primary molecular cause 2013). Telomeres are bound by a specialized complex known of aging. Short telomeres block the proliferative capacity as shelterin that has crucial functions in telomere length regu- of stem cells, affecting their potential to regenerate tissues, lation and in the protection of telomeres from the DNA dam- and trigger the development of age-associated diseases. age response (DDR) by masking the chromosome ends from the DNA repair machinery through repression of the ATM and Mutations in telomere maintenance genes are associated ATR signaling pathways (Palm and de Lange, 2008; Fig. 1 C). with pathologies referred to as telomere syndromes, in- The shelterin complex is composed of six proteins: telomeric cluding Hoyeraal-Hreidarsson syndrome, dyskeratosis repeat binding factors 1 and 2 (TRF1 and TRF2), TRF1- interacting protein 2 (TIN2), protection of telomeres protein 1 congenita, pulmonary fibrosis, aplastic anemia, and liver (POT1), TIN2, POT1-interacting protein (TPP1), and repressor/ fibrosis. Telomere shortening induces chromosomal insta- activator protein 1 (RAP1; Fig. 1 C; de Lange, 2005; Martínez bility that, in the absence of functional tumor suppressor and Blasco, 2010, 2011). genes, can contribute to tumorigenesis. In addition, muta- Telomeres shorten with each cell division as a result of the incomplete replication of linear DNA molecules by conven- tions in telomere length maintenance genes and in shel- tional DNA polymerases, which is called the end-replication terin components, the protein complex that protects problem (Watson, 1972; Olovnikov, 1973). Telomerase com- telomeres, have been found to be associated with differ- pensates for telomere attrition through de novo addition of TTA​ ent types of cancer. These observations have encouraged GGG repeats onto chromosome ends in those cells where it is normally expressed, such as pluripotent stem cells and adult the development of therapeutic strategies to treat and pre- stem cell compartments (Liu et al., 2007; Flores et al., 2008; vent telomere-associated diseases, namely aging-related Marion et al., 2009). Telomerase is composed of a reverse tran- diseases, including cancer. Here we review the molecular scriptase subunit (TERT) as well as an associated RNA compo- nent (Terc), which is used as a template for the de novo addition mechanisms underlying telomere-driven diseases and of telomeric repeats (Fig. 1 C; Greider and Blackburn, 1985). highlight recent advances in the preclinical development Although telomerase is expressed in adult stem cell compart- of telomere-targeted therapies using mouse models. ments, this is not sufficient to counteract telomere attrition associated with cell division throughout life, and therefore telo- Telomeres, telomerase, and shelterins meres shorten with age in vitro and in vivo (Harley et al., 1990; THE JOURNAL OF CELL BIOLOGY CELL OF JOURNAL THE Telomeres form a special heterochromatic structure at the end Hastie et al., 1990; Lindsey et al., 1991; Collado et al., 2007; of linear chromosomes that protects them from degradation and Liu et al., 2007; Flores et al., 2008; Marion et al., 2009). This DNA repair and recombination activities. Thus, telomeres are progressive telomere shortening eventually leads to critically essential to ensure chromosome stability (Blasco, 2005; Palm short telomeres that can impair the regenerative capacity of tis- and de Lange, 2008). Mammalian telomeres comprise several sues and has been proposed as one of the molecular hallmarks kilobases, between 10 and 15 kb in humans and 25 and 50 kb in of aging (López-Otín et al., 2013). In mice, it has been shown mice, of tandem TTA​GGG DNA repeats (Blasco, 2005). Telo- that the rate of increase in the percentage of short telomeres, meres are characterized by the presence of a 30–400-nucleo- rather than the rate of telomere shortening throughout life, is a tide-long 3′ overhang of a G-rich strand, known as the G-strand significant predictor of life span (Vera et al., 2012). Shortened overhang. The G-strand overhang can fold back and invade the telomeres induce a DDR that leads to a growth arrest, during double-stranded telomeric region, forming the so-called T-loop which cells attempt to repair the damage and, if DNA damage and generating a displacement loop, or D-loop. The T-loop is irreparable, triggers replicative senescence (Zou et al., 2004; Fumagalli et al., 2012). Senescent cells progressively accumu- Correspondence to Maria A. Blasco: [email protected] Abbreviations used: 6-thio-dG, 6-thio-2 -deoxyguanosine; AAV, adeno-associ- ′ © 2017 Martínez and Blasco This article is distributed under the terms of an Attribution– ated vector; ALT, alternative lengthening of telomeres; BM, bone marrow; DC, Noncommercial–Share Alike–No Mirror Sites license for the first six months after the dyskeratosis congenita; DDR, DNA damage response; HHS, Hoyeraal-Hreidars- publication date (see http​://www​.rupress​.org​/terms​/). After six months it is available under son syndrome; HSPC, hematopoietic stem/progenitor cell; IPF, idiopathic PF; a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International MC, mitotic catastrophe; PF, pulmonary fibrosis; TPE, telomere position effect. license, as described at https​://creativecommons​.org​/licenses​/by​-nc​-sa​/4​.0​/).

The Rockefeller University Press $30.00 J. Cell Biol. Vol. 216 No. 4 875–887 https://doi.org/10.1083/jcb.201610111 JCB 875 Figure 1. The shelterin complex and the structure of telomeres. (A) Representative image of a metaphasic chromosome stained with DAPI (blue) and the telomeric DNA–specific peptide nucleic acid probe (yellow). (B) Schematic model of the shelterin complex bound to a telomere in a T-loop configuration. Telomeres contain a double-stranded region of TTA​GGG repeats and a 150–200-nucleotide-long single-stranded DNA overhang of a G-rich strand. The G-strand overhang (gray) invades the telomeric double-stranded DNA region to form a protective T-loop, with a displacement D-loop at the invasion site. (C) Schematic representation of telomere-bound proteins, the shelterin complex, and telomerase. The shelterin complex is composed of the telomeric repeat binding factor 1 (TRF1, also known as TERF1), TRF2 (also known as TERF2), repressor-activator protein 1 (RAP1, also known as TERF2IP1), POT1-TIN2 organizing protein (TPP1, also known as ACD), TIN2 (also known as TIFN2), and protection of telomeres protein 1 (POT1). TRF1, TRF2, and POT1 bind directly to telomeric DNA repeats, with TRF1 and TRF2 binding to telomeric double-stranded DNA and POT1 to the 3′ single-stranded G-overhang. TIN2 binds TRF1 and TRF2 through independent domains and recruits the TPP1–POT1 complex, constituting the bridge among the different shelterin components. Telomerase is a two-partner enzyme, the catalytic subunit (TERT) and the RNA template (TERC), that recognizes the 3′-OH at the end of the G-strand over- hang and elongates the telomere.

late during life and secrete factors that influence age-associated Lee et al., 1998; Herrera et al., 1999; Leri et al., 2003; Ferrón et diseases (Tchkonia et al., 2013). Indeed, senescence has been al., 2004). Telomere shortening with age has also been observed proposed as a mechanism that evolved to protect from cancer, in several mouse stem cell compartments from different tissues with the drawback of promoting age-associated diseases (Shay, independently of high or low tissue proliferation rates (Flores 2016). Therapeutic interventions based on either chemical acti- et al., 2008). Telomerase-deficient mice show a worsening of vators of telomerase or telomerase-based gene therapy are cur- phenotypes with increasing mouse generations owing to inher- rently being investigated in mouse models for their potential to ited progressively shorter telomeres, with the later generations improve health and extend life span, and as a treatment for short showing severe phenotypes and premature death at pre-repro- telomere syndromes (Bernardes de Jesus et al., 2011; Bär et al., ductive ages (Lee et al., 1998; Herrera et al., 1999; García-Cao 2014, 2015; Bär and Blasco, 2016). et al., 2002). This phenomenon first indicated genetic antici- pation associated with telomerase deficiency. However, and in Aging-associated diseases and support of critical telomere shortening being a determinant of telomeropathies aging and longevity, increased TERT expression in the context Telomeres shorten progressively throughout an individual’s life- of cancer-resistant transgenic mice was sufficient to delay aging time (Cawthon et al., 2003; López-Otín et al., 2013; Sanders and and extend mouse longevity by 40% (Tomás-Loba et al., 2008). Newman, 2013). Telomere attrition in stem cell compartments More recently, these findings have been translated into a po- impairs their tissue and self-renewal capacity and is considered tential therapeutic strategy by using adeno-associated vectors to be one of the primary molecular causes of aging and the onset (AAVs) to transiently activate telomerase in adult tissues (Ber- of aging-associated diseases (Flores et al., 2005; Sharpless and nardes de Jesus et al., 2012; Bär et al., 2014). Telomeropathies DePinho, 2007). The first demonstration that telomere shorten- or telomere syndromes develop when telomere attrition occurs ing is at the origin of age-related pathologies and is a determi- prematurely as a consequence of germline mutations in genes nant of longevity came from the study of telomerase-deficient coding for factors involved in telomere maintenance and repair mice, which show premature aging phenotypes in both low- (Table 1). Human telomeropathies are mainly associated with proliferating (heart, brain) and high-proliferating compartments Hoyeraal-Hreidarsson syndrome (HHS), dyskeratosis congen- (bone marrow [BM], gut, skin, and testis; Blasco et al., 1997; ita (DC), aplastic anemia, and pulmonary and liver disease and

876 JCB • Volume 216 • Number 4 • 2017 Clinical features of telomeropathies

Aplastic anemia: Aplastic anemia is a BM failure state characterized by low blood counts and an insufficiency of hematopoietic cells in the BM (Fogarty et al., 2003). Idiopathic pulmonary fibrosis (IPF): IPF is characterized by cough, dyspnea, impaired gas exchange, and reduced lung volume. Pathologically, there is patchy fibrosis of the lungs and interstitial inflammation, normal lung alternating with fibrosis, inflammation, and collagen deposition (Armanios et al., 2007). Liver disease: Liver disease associated with telomere shortening consists of mainly fibrosis with inflammation and nodular regenerative hyperplasia, a lead- ing cause of noncirrhotic portal hypertension (Calado et al., 2009a). Dyskeratosis congenita (DC): DC is classically diagnosed by the presence of the mucocutaneous triad of nail dysplasia, skin pigmentation, and oral leuko- plakia or the presence of one feature of the triad in combination with BM failure and two other DC-associated findings. Patients with DC are at very high risk of BM failure, PF, liver fibrosis, stenosis, premature hair graying, leukemia, and squamous cell cancer of the head, neck, or anogenital regions (Ballew and Savage, 2013). Hoyeraal-Hreidarsson syndrome (HHS): HHS is a multisystem genetic disorder characterized by very short telomeres (less than first percentile for age) and is considered a clinically severe variant of DC. Patients with HHS present in early childhood with cerebellar hypoplasia, microcephaly, immunodeficiency, BM failure, and intrauterine growth retardation. The DC-associated mucocutaneous triad is also a trait of HHS (Glousker et al., 2015). are nowadays considered more as a spectrum disorder than dis- encoding a toxic double-homeobox protein, has been linked to tinct diseases (see Clinical features of telomeropathies; Holo- facioscapulohumeral muscular dystrophy, a disease presenting han et al., 2014; Stanley and Armanios, 2015). Although these with progressive atrophy and weakness of the facial, scapular, diseases show a wide and complex range of clinical symptoms, and upper arm muscles (Stadler et al., 2013). This disease has all of them are characterized by presenting with critically short highly variable clinical manifestation, and delayed onset that telomeres. The age of onset and the severity of clinical manifes- can be explained by age-associated telomere shortening (Stadler tations vary among individuals. These syndromes are character- et al., 2013). Thus, facioscapulohumeral muscular dystrophy ized by premature loss of the regenerative capacity of tissues, constitutes the first human disease to demonstrate a potential affecting tissues with both high and low proliferation rates (Ar- contribution of TPE to the age-associated diseases. manios and Blackburn, 2012; Holohan et al., 2014). As in mice, disease anticipation is also found in human families with telo- Mouse models for PF, aplastic anemia, mere syndromes, with the mutation generally first manifesting DC, and HHS in adults with pulmonary fibrosis (PF), and the more severe phe- The study of mouse models genetically deficient in telomerase notypes appearing in pediatric populations (immunodeficiency) and telomeric proteins has been crucial to understanding the role and young adults (aplastic anemia) from the next generations of telomere biology in human telomeropathies. In particular, (Armanios and Blackburn, 2012). the telomerase knockout, Pot1 knockout, and Trf1 conditional In addition to telomere-mediated replicative senescence, knockout mouse models have recently served as proofs-of-prin- telomere length can have an impact on human diseases by ciple of the causal role of DNA damage stemming from dys- regulating gene expression through telomere position effects functional telomeres in DC, idiopathic PF (IPF), and aplastic (TPEs). TPEs involve the spreading of telomeric heterochroma- anemia (see text box; Hockemeyer et al., 2008; He et al., 2009; tin to silence nearby genes. In human cells, it has been shown Martínez et al., 2009b; Beier et al., 2012). Recently, mutant that gene expression is affected by telomere length (Stadler et mice expressing p53 lacking the C-terminal domain, a protein al., 2013; Robin et al., 2014). In particular, genes located near that is not directly involved in telomere maintenance, have been the telomere at chromosome 4q are progressively up-regulated shown to be remarkable models for human telomeropathies, in with decreasing telomere length, and this occurs long before particular for HHS (see text box; Simeonova et al., 2013). terminal telomere shortening would induce replicative senes- cence (Stadler et al., 2013). Interestingly, among the genes an- Aplastic anemia mouse models alyzed, the effect of TPE is most prominent with DUX4, the The aplastic anemia phenotypes in mouse (see text box) are one located nearest to the 4q telomere. Expression of DUX4, provoked either by partially depleting TRF1 in the BM stem

Table 1. Genes known to cause telomeropathies when defective, as well as the telomere-related processes and protein complexes in which they are involved

Process/protein complex Genes Telomeropathies

Telomerase core components TERC DC, IPF, aplastic anemia, liver disease TERT DC, HHS, IPF, aplastic anemia, liver disease Telomerase biogenesis DKC1 DC, HHS, IPF, aplastic anemia NOP10 DC, IPF, aplastic anemia NHP2 DC, IPF, aplastic anemia Telomerase trafficking TCBA1 DC Shelterin components TIN2 DC, HHS TPP1 DC, HHS, aplastic anemia Telomeric DNA synthesis RTEL1 HHS, IPF CTC1 DC TERC RNA processing PARN DC, IPF NAF1 IPF

The diseases found associated with the mutated genes are indicated (Calado et al., 2009a; Holohan et al., 2014; Glousker et al., 2015; Stanley and Armanios, 2015; Stanley et al., 2016).

Telomeres as therapeutic targets • Martínez and Blasco 877 cell and progenitor compartments (hematopoietic stem/pro- dromes (Savage et al., 2008; Alder et al., 2015b; Hoffman et al., genitor cells [HSPCs]) or by transplanting irradiated wild- 2016). Interestingly, a TIN2 mutation in its TRF1-interacting type mice with BM from late-generation telomerase-deficient domain has recently been identified in a patient with sporadic Tert knockout mice presenting with short telomeres (Beier et PF who showed normal telomere length in the peripheral blood al., 2012; Bär and Blasco, 2016). Partial depletion of the Trf1 compartment, underlining the importance of telomere protec- gene in the BM causes a persistent DDR at telomeres that leads tion proteins and not only telomere length in human telomere to a fast clearance of those HSPCs lacking TRF1. Compensa- syndromes (Hoffman et al., 2016). tory hyperproliferation of the remaining HSPCs to regenerate the BM results in a concomitant rapid attrition of telomeres DC and HHS mouse models (Beier et al., 2012). The short telomeres in BM of both mouse DC and HHS share a common etiology, are typically caused by models faithfully recapitulate human acquired and congenital germline mutations in telomere biology genes, and are charac- forms of aplastic anemia characterized by peripheral pancyto- terized by very short telomeres (see text box; Glousker et al., penia and marrow hypoplasia. 2015). In mice, double deletion of Pot1b and Terc gives rise to some DC features such as hyperpigmentation and BM failure PF mouse models (Hockemeyer et al., 2008; He et al., 2009). HHS is considered Among the telomere syndromes, IPF is the most common con- a clinically severe variant of DC (Glousker et al., 2015). Telo- dition associated with telomere dysfunction in humans (see meres in HHS patients are shorter than those of age-matched text box; Armanios and Blackburn, 2012). Mutations in telo- patients with classic DC (Alter et al., 2012). Although exceed- mere genes have been found in up to 25% of familial and 1–3% ingly short telomeres are considered the main cause of HHS, of sporadic IPF patients (Armanios et al., 2007; Alder et al., it is likely that other telomeric or nontelomeric defects also 2008; Cogan et al., 2015; Kannengiesser et al., 2015; Stuart et contribute to the pathology of HHS, perhaps distinguishing it al., 2015; Stanley et al., 2016). Sporadic cases of IPF, not as- from DC (Glousker et al., 2015). Indeed, to our knowledge, the sociated with mutations in telomere maintenance genes, also only mouse mutant that faithfully models human HHS does not show shorter telomeres compared with age-matched controls, harbor mutations in any of the known telomere maintenance with 10% of the patients showing telomeres as short as those genes associated with these diseases, but rather a mutation in of the telomerase mutation carriers (Alder et al., 2008). In spite TP53 gene (Simeonova et al., 2013). p53Δ31/Δ31 mice, homozy- of PF and emphysema being the most frequent manifestations gous mutant mice expressing a truncated p53 protein lacking of telomere defects in humans, telomerase-deficient mice with the C-terminal domain, suffer from aplastic anemia and PF as critically short telomeres do not spontaneously develop pul- well as other phenotypic traits associated with DC and its severe monary disease. Telomere dysfunction in telomerase-deficient variant, HHS (Simeonova et al., 2013). Thus, p53Δ31/Δ31 mice are mice induces senescence in alveolar progenitor cells and re- born at the expected Mendelian ratio, but most die 14–43 d after capitulates the regenerative defects, inflammatory responses, birth because of aplastic anemia and heart failure, develop PF, and susceptibility to injury that are characteristic of human and present cutaneous hyperpigmentation, nail dystrophy, and telomere-mediated lung disease, although no IPF was reported oral leukoplakia as in DC. In addition, some mice also present in these mouse models (Alder et al., 2015a). Exposure to ciga- HHS features such as small size, hypogonadism, and cerebel- rette smoke, which is known to accelerate PF onset in humans, lar hypoplasia. The p53Δ31 protein shows increased p53 activity induces emphysema but not PF in telomerase-deficient mice leading to down-regulation of genes involved in telomere me- (Alder et al., 2011). These findings suggested that additional tabolism such as Dkc1, Rtel1, Tin2, and Trf1, thereby explaining damages, or a telomere damage of higher severity, might be re- the appearance of phenotypes related to human telomere syn- quired for PF onset in mice. This was demonstrated in work dromes (Simeonova et al., 2013). from our laboratory, whereby we generated two independent mouse models that develop PF. In one of them, treatment of Therapeutic strategies in the treatment of late-generation telomerase-deficient mice presenting short telo- telomere syndromes meres with low doses of bleomycin, which normally does not Despite the growing understanding of the molecular defects as- lead to PF in wild-type mice, results in full-blown progressive sociated with telomeropathies, the only current interventions to PF in telomerase-deficient mice (Povedano et al., 2015). In the treat these diseases involve organ transplantation; i.e., BM, liver, second mouse model, severe PF was developed by induction and lung (Holohan et al., 2014; Stanley and Armanios, 2015). of telomere dysfunction in the absence of telomere shortening Although organ transplantation improves the physical condi- specifically in the lungs, by genetically deleting the shelterin tion of patients, it does not address the underlying cause of the component Trf1 from epithelial type II alveolar cells (Pove- symptoms, which is short telomeres. Patients diagnosed with dano et al., 2015). In both models, we observed activation of interstitial lung disease presenting with short telomeres also the p53/p21 and p19ARF pathways, resulting in cell senes- showed subclinical BM and liver abnormalities, in some cases cence and apoptosis. Both TERT and TRF1 deficiencies were in the absence of clinical manifestations in peripheral blood previously shown to lead to a decreased ability of stem cells to count and liver dysfunction (George et al., 2015). These ob- regenerate tissues (Flores et al., 2005; Schneider et al., 2013). servations clearly favor a telomerase-activating therapy against Thus, increased lung cell loss associated with severe telomere other alternatives, such as organ transplant for the treatment of dysfunction triggers aberrant lung healing by fibroblasts, even- telomeropathies, because it also addresses the molecular defects tually leading to scar formation and PF. We showed that a DNA- of other affected organs. Indeed, the use of telomerase activators damaged burden above a certain threshold, combined with de- in the treatment of aging-associated conditions has raised com- fective stem cell regeneration ability, lead to the development of mercial interest. Among telomerase chemical activators, TA- PF (Povedano et al., 2015). The TRF1-interacting shelterin pro- 65, a small molecule derived from Astragalus membranaceus tein TIN2 has also been found to be mutated in telomere syn- extracts, is the most widely studied. The telomerase activator

878 JCB • Volume 216 • Number 4 • 2017 Figure 2. Natural factors and therapeutic interventions affecting telomere-mediated diseases. Telomere shortening naturally occurs as a consequence of cell division throughout life, whose pace can be influenced by genetic and environmental factors. Shortened unprotected telomeres elicit a DDR that induces cellular senescence, impacting the regenerative capacity of tissues and giving rise to a whole range of age-associated diseases as well as the so-called telomeropathies, in which tissue degeneration occurs prematurely as a consequence of inherited defects in telomere maintenance. Several therapeutic interventions are being assessed to counteract telomere shortening: among others, chemical activators of telomerase (TA-65), activators of the telomerase reverse transcription (TERT) transcription (sex hormones), intracellular administration of TERT mRNA, and telomerase gene therapy (AAV9-TERT). Sponta- neous mutations that activate telomerase expression or ALT result in telomere lengthening that, if occurring in genetically unstable checkpoint deficient cells, allows them to divide unlimitedly and eventually become cancer cells. Several therapeutic strategies based on chemical induction of telomere dysfunction have been assessed as anticancer therapies. Among others, we highlight the use of a chemical inhibitor of telomerase (imetelstat), a nucleoside analogue (6-thio-dG), and the use of molecules that inhibit shelterin components.

TA-65 has been shown to lead to moderate telomere lengthen- farct treatment led to lower fibrotic scarring of the heart and ing and improvement of some aging-related parameters in mice increased cardiac myocyte proliferation concomitant with tran- and humans, although no effect on longevity has been observed scriptional changes suggestive of a regenerative signature (Bär (Fig. 2; Bernardes de Jesus et al., 2011; Harley et al., 2013). In et al., 2014). These findings support the notion that telomere addition, androgen therapy has been applied as a treatment for shortening is at the origin of age-related diseases and that, by aplastic anemia for many years without knowing its mechanism reverting this process with telomerase expression, it is possible of action (Fig. 2; Shahidi and Diamond, 1961; Jaime-Pérez et to delay and more effectively treat age-associated pathologies, al., 2011). It was subsequently reported that sex hormones ac- such as cardiovascular diseases. In addition, TERT gene ther- tivate TERT transcription (Calado et al., 2009b). Indeed, tes- apy is particularly attractive for the clinical treatment of human tosterone therapy in mice suffering from aplastic anemia was telomere syndromes associated with telomerase mutations and shown to up-regulate telomerase expression, rescue telomere short telomeres. Indeed, by using the mouse preclinical model attrition, and extend the life span of these mice (Fig. 2; Bär et of aplastic anemia provoked by short telomeres in the BM, we al., 2015). In humans, administration of a synthetic androgen, demonstrate that AVV9-Tert rescues aplastic anemia and mouse danazol, to patients with telomeropathies resulted in telomere survival by inducing telomere lengthening in peripheral blood elongation in circulating leukocytes in association with hema- and BM cells as well as increasing blood counts (Bär et al., tologic improvement (Townsley et al., 2016). However, endog- 2016). We are currently investigating the feasibility of AVV9- enous telomerase reactivation strategies are applicable only in Tert therapy in the treatment of PF, with positive results so far those clinical cases not associated with mutations in telomere (unpublished data). An alternative nonintegrative method to maintenance genes such as TERT or TERC. In this regard, ther- transiently express TERT based on TERT mRNA delivery into apeutic interventions based on telomerase-based gene therapy human cells in culture has been developed, although it has not are currently being investigated in mouse models for their po- yet been tested in vivo (Fig. 2; Ramunas et al., 2015). tential to improve health and extend life span, and as a treatment Telomerase activation strategies should be taken with cau- for short telomere syndromes (Bär and Blasco, 2016). tion given their potential off-target effects and their ability to We have developed a therapeutic strategy by using AAVs promote cancer (Bär and Blasco, 2016). Nevertheless, recent to transiently activate telomerase in adult tissues (Fig. 2; Ber- data from our group demonstrated that chimeric mice pre- nardes de Jesus et al., 2012; Bär et al., 2014). Treatment with senting hyperlong telomeres showed neither disadvantageous Tert gene therapy using nonintegrative, replication-incompe- effects nor enhanced predisposition for cancer development tent AAV9 vectors of adult mice was able to delay aging and compared with control chimeric mice presenting normal telo- increase longevity by decreasing age-related pathologies such mere length, indicating that long telomeres do not cause nega- as osteoporosis and glucose intolerance, as well as neuromus- tive consequences to the organism (Varela et al., 2016). Indeed, cular and cognitive decline. Furthermore, the onset of cancer in chimeric mice with hyperlong telomeres, cells in compart- was delayed in the TERT-treated mice (Bernardes de Jesus et ments with high rates of renewal retain longer telomeres and al., 2012). Interestingly, AAV9-Tert delivery specifically to the accumulate fewer short telomeres, less DNA damage burden, heart was sufficient to significantly increase mouse survival and and lower levels of p53 with age (Varela et al., 2016). Thus, up heart function upon myocardial infarction. AAV9-Tert after in- to date experimental data in preclinical settings provide proof

Telomeres as therapeutic targets • Martínez and Blasco 879 of concept for the viability of telomerase activation strategies constitutes the second blockade against transformation in cells to counteract telomere shortening and its associated conse- that bypassed the first barrier of senescence (Fig. 3 D). Reac- quences. In particular, the use of TERT gene therapy consti- tivation of either telomerase activity or the alternative length- tutes a promising candidate in the prevention and treatment ening of telomeres (ALT) allows these premalignant cells to of human telomeropathies mediated by TERT mutations and escape crisis, undergo unlimited divisions (immortalization), deserves further research efforts for clinical implementation. and evolve into a fully transformed cancerous state (Fig. 3 E; It should be noted that TERT gene therapy in the treatment of Pickett and Reddel, 2015; Shay, 2016). Indeed, ∼90% of human telomeropathies mediated by mutations in other genes associ- cancers present telomerase activity (Kim et al., 1994). To date, ated with telomere metabolism (TERC, DKC1, NOP10, NHP2, several mechanisms underlying reactivation of telomerase in TCAB1, TIN2, TPP1, RTEL1, CTC1, PARN, and NAF1) may, cancer cells have been described: notably, mutations in TERT however, not be effective. promoter, alterations in the alternative splicing of TERT pre- mRNA, gene amplification, epigenetic changes, alterations in Telomeres and malignant transformation regulatory factors, and /or disruption of TPE (Jafri et al., 2016). Telomere shortening throughout life is a natural consequence of Whether telomerase reactivation is an early or late event during cell division because of the end replication problem, replication tumorigenesis is still a debated question. Recent data show- fork collapse, oxidative stress, and nucleolytic processing. Crit- ing that TERT promoter mutations are found at early stages ically shortened telomeres trigger replicative senescence that of carcinogenesis in most tumor types support the hypothesis causes stem cell dysfunction and inflammation, in turn causing that telomerase reactivation occurs at early time points during aging-associated diseases (Fig. 3 A; Jafri et al., 2016). In the neoplastic transformation (Kinde et al., 2013; Allory et al., situation of acquired germline mutations in genes coding for 2014; Hurst et al., 2014; Baerlocher et al., 2015; Chan et al., factors involved in telomere maintenance and repair, telomere 2015; Huang et al., 2015; Tefferi et al., 2015; Jafri et al., 2016). shortening is accelerated, and replicative senescence occurs However, it is still under debate whether telomerase or ALT re- prematurely, giving rise to telomeropathies (Fig. 3 B; Holohan activation is the driver or simply a facilitator of postcrisis cell et al., 2014). In mammalian cells, replicative senescence can be growth. It is possible that other molecular events may be re- bypassed by acquisition of loss-of-function mutations in tumor quired to facilitate the escape from crisis. Studies on telomere suppressor genes that permit further proliferation during a pe- dynamics and karyotype analyses in a broad range of human riod of time that has been named the extended life span period tumor types at early stages underpin telomere crisis as a key (Fig. 3 C; Wright and Shay, 1992; Shay and Wright, 2005; Shay, event driving genomic instability and clonal evolution during 2016). Proliferation of premalignant cells during the extended progression to malignancy (Shih et al., 2001; Meeker et al., life span period results in further telomere shortening. Even- 2004; Lin et al., 2010; Davoli and de Lange, 2011; Roger et tually, cells enter the so-called crisis state, during which short al., 2013). Fused chromosomes in postcrisis cells lead to large- telomeres contribute to genomic instability. Recent work has scale genomic rearrangements through breakage-fusion-bridges unraveled how dysfunctional telomeres govern cell fate during cycles (B-F-B), aneuploidy, tetraploidization, nonreciprocal senescence, crisis, and transformation in human cells. Replica- translocations, chromothripsis, and kataegis, which promote ac- tive senescence is triggered when at least five telomeres become quisition of oncogenic mutations and malignant traits required dysfunctional, a critical damage threshold to elicit a DDR char- for a fully malignant phenotype (Artandi and DePinho, 2010; acterized by p53 activation (Fig. 3, A and B; Kaul et al., 2011). Martínez and Blasco, 2010; Davoli and de Lange, 2012; Ma- At that point, named the intermediate telomere state, telomeres ciejowski et al., 2015). are too short to be fully functional and are presumably unable to form the T-loop structure but retain sufficient protective shel- Mouse models for telomere-driven cancer terin to inhibit end-to-end fusions (Cesare et al., 2009, 2013; development Kaul et al., 2011; Cesare and Karlseder, 2012). Upon loss of In vivo studies with transgenic mouse models of telomere dys- p53 and retinoblastoma protein (Rb), cells bypass senescence function in combination with loss-of-function mutations in and continue to grow despite having more than five G1-phase tumor suppressor genes strongly underscore the statement that intermediate state telomeres, experiencing further telomere telomere dysfunction leads to genomic rearrangements that shortening and entering the uncapped state, when they do not are permissive for cancer initiation and progression (Table 2; retain any protective properties and fuse (Fig. 3 C). A few fused Martínez and Blasco, 2011). Thus, mice doubly deficient for telomeres is sufficient to lead to spindle assembly checkpoint– p53 and telomerase, the Terc −/− p53−/− mouse model, present a independent mitotic arrest, during which telomere dysfunction reduced tumor latency compared with single p53−/− mice, and is amplified through Aurora B–dependent removal of TRF2, Terc −/− p53+/− mice are prone to develop epithelial cancers typ- causing cell death during crisis, a second proliferative barrier ically observed in older humans (Chin et al., 1999). In addi- (Fig. 3 D; Hayashi et al., 2012, 2015). Cell death in mitosis, also tion, deficiency in the mismatch repair protein MSH2 abolishes known as mitotic catastrophe (MC), occurs as a consequence the tumor-suppressor effects of short telomeres and prevents of failure to complete mitosis. MC is driven by a complex and degenerative pathologies in Terc −/− Msh2−/− mice (Martinez et poorly understood signaling cascade that antecedes apoptosis, al., 2009a). Similarly, and despite the fact that constitutive de- necrosis, senescence, or autophagy (Levine and Kroemer, 2008; letion of either Trf1 or Pot1a results in embryonic lethality, tis- Vitale et al., 2011). The molecular mechanisms that underlie sue-specific conditional deletion of these genes in combination MC, especially those that sense mitotic failure to engage the with p53-null mutation leads to tumorigenesis (Martínez et al., apoptotic, autophagy, or necrotic machinery, remain to be elu- 2009b; Pinzaru et al., 2016). Thus, Trf1lox/lox K5-cre p53−/− mice cidated. It is tempting to speculate that structural and epigenetic lacking TRF1 in stratified epithelia spontaneously develop inva- changes experienced by multicentric chromosomes could serve sive and genetically unstable squamous cell carcinomas in the as the signal for MC induction. Cell death during crisis thereby tail and ear skin (Martínez et al., 2009b). Depletion of POT1a

880 JCB • Volume 216 • Number 4 • 2017 Figure 3. Impact of telomere shortening on aging-associated diseases, telomeropathies, and cancer. (A) Young, healthy cells contain long, fully protected telomeres that progressively shorten with increased cell divisions because of the end replication problem, replication fork collapse, nucleolytic processing, and oxidative stress. This progressive telomere shortening eventually leads to some critically short deprotected telomeres that have been termed interme- diate-state telomeres. In this intermediate state of deprotection, telomeres retain enough shelterin to inhibit fusions but induce a DDR characterized by formation of the so-called telomere-induced focus (TIF). Replicative senescence is triggered when at least five telomeres become dysfunctional (more than five telomere-induced focuses), a critical damage threshold to elicit a DDR characterized by p53 activation. Telomere attrition in stem cell compartments impairs their tissue and self-renewal capacity and is considered to be one of the primary molecular causes of aging and the onset of aging-associated diseases. (B) Telomeropathies or telomere syndromes develop when telomere attrition occurs prematurely as a consequence of germline mutations in genes coding for factors involved in telomere maintenance and repair. Successive telomere shortening across generations exhibits genetic anticipation, whereby diseases show a progressively earlier age of onset and an aggravation of symptoms. (C) Senescence can be bypassed by acquisition of loss-of-function mutations in p53 and p16/Rb tumor suppressor genes that permit further proliferation during a period of time that has been named the extended life span period, during which cells experience further telomere shortening, eventually entering the uncapped state, when they do not retain any protective properties and

Telomeres as therapeutic targets • Martínez and Blasco 881 and p53 in common lymphoid progenitor cells, Pot1alox/lox for IPSC generation and the maintenance of pluripotency, the p53lox/lox hCD2-iCre mice, induces genetic instability, acceler- mechanisms by which TRF1 impacts stemness and carcinogen- ates the onset, and increases the severity of T cell lymphomas esis have yet to be elucidated. It is tempting to speculate that (Table 2; Pinzaru et al., 2016). Similarly, simultaneous inacti- TRF1 favors the proliferative capacity of stem and cancer cells. vation of Pot1a and p53 in endometrial epithelium, Pot1alox/lox Mutations in the shelterin component POT1 that disrupt p53lox/lox Sprr2f-Cre mice, results in invasive metastatic endome- proper telomere capping and result in telomere aberrations have trial adenocarcinomas presenting nuclear atypia and tetraploid been linked to familial melanoma, familial glioma, Li-Frau- genomes (Akbay et al., 2013). In line with defects in shelterin meni–like syndrome, mantle cell lymphoma, parathyroid ade- components as drivers of genome instability, mice harboring a noma, and chronic lymphocytic leukemia (Newey et al., 2012; hypomorphic mutation in TPP1 in a p53−/− background show Ramsay et al., 2013; Bainbridge et al., 2014; Robles-Espinoza increased carcinomas (Else et al., 2009). Interestingly, complete et al., 2014; Shi et al., 2014; Zhang et al., 2014; Calvete et al., TPP1 and p53 abrogation in stratified epithelia, the Tpp1lox/lox 2015). Importantly, all the identified POT1 variants conferred a K5-cre p53−/− mouse model, does not lead to epithelial carcino- telomere-lengthening effect, suggesting that telomere elongation mas, most likely because TPP1 is required to recruit telomerase in combination with the telomere aberrations induced by these to chromosome ends, mimicking the tumor-resistant phenotype mutations favors tumor development. Introduction of POT1 of telomerase-deficient mice (Table 2; Tejera et al., 2010). mutations found in cutaneous T cell lymphoma in human and It should be noted that similar mutations in a different mouse cells showed that inhibition of POT1 results in defective genetic context give rise to opposing outcomes resulting in telomere replication caused by impaired CST (CTC1-STN1- cancer phenotype suppression. For example, late generation of TEN1) function at telomeres and activates ATR-dependent mice doubly deficient for telomerase and the tumor suppres- DNA damage signaling (Pinzaru et al., 2016). Attenuation of sor INK4a/ARF, Terc −/− Ink4a/Arf−/− mice, show a cancer sup- the ATR kinase pathway allows cancer cells lacking POT1 to pression phenotype and increase survival (Khoo et al., 2007). proliferate, suggesting that POT1-mediated tumorigenesis may Trf1lox/lox p53−/− K-RasLSLG12V mice constitute an example of be accompanied by concomitant compensatory mechanisms shelterin depletion as a cancer protective mechanism. Thus, si- that down-regulate the ATR-dependent DDR (Pinzaru et al., multaneous expression of K-RASG12V and TRF1 depletion upon 2016). It is intriguing that specific POT1 variants give rise to Cre recombinase expression in the lungs impairs the growth of different cancer types. Future work warrants elucidation of the lung carcinomas and increases mouse survival independently molecular mechanisms that determine the specificity of certain of telomere length (Table 2; García-Beccaria et al., 2015). specific disease-associated mutations in shelterin components. These studies clearly revealed that the neoplastic outcome of Mutations in TPP1 and RAP1 have also been found to be telomere dysfunction depends on the status of the DNA dam- associated with familial melanoma (Aoude et al., 2014). In ad- age surveillance mechanisms, acting oncogenes, as well as on dition, families carrying TPP1 and RAP1 mutations were en- tissue-specific factors. riched with other cancer types, suggesting that these variants also cause predisposition to a broader spectrum of tumors than Role of shelterins in human cancer just melanoma. Although the telomeric phenotypes induced development and progression by these mutations have not been addressed, the observation Mutations in shelterin components have also been found in can- that four of the five mutations found inTPP1 clustered in the cer. Several studies have reported up-regulation of the shelterin POT1-binding domain suggests that POT1 recruitment to telo- complex TRF1 and TRF2 in lung, gastric, breast, and renal can- meres is disrupted, thereby causing similar phenotypes as ob- cers, suggesting that their overexpression might confer prolifer- served in POT1 variants discussed earlier (Liu et al., 2004). More ative advantages to tumor cells (Miyachi et al., 2002; Saito et intriguing is the finding of RAP1 variants co-segregating with al., 2002; Nakanishi et al., 2003; Diehl et al., 2011; Pal et al., melanoma because RAP1 is dispensable for telomere function 2015). However, the role of TRF1 and TRF2 in cancer develop- (Martinez et al., 2010; Sfeir et al., 2010). A possible explanation ment and progression is still unknown. One possible explana- for the role of RAP1 in cancer could be either through its role in tion could be that high level of TRF2 in tumor cells decreases repressing homology-directed repair at telomeres (Martinez et their ability to recruit and activate natural killer cells, thereby al., 2010; Sfeir et al., 2010) or through its role in telomere main- contributing to the bypass of innate immunosurveillance (Bi- tenance in the absence of telomerase (Martínez et al., 2016). roccio et al., 2013). Interestingly, TRF1 is highly expressed in pluripotent stem cells and adult stem cells (Boué et al., 2010; Telomeres as anticancer targets Varela et al., 2011; Schneider et al., 2013). TRF1 is essential for Given that most tumors (85–90%) present telomerase activity, both induction and maintenance of pluripotency in a telomere whereas telomerase is absent in most normal tissues, or is strictly length–independent manner (Schneider et al., 2013). TRF1 is a regulated in transient-amplifying stem cells, inhibition of telo- direct transcriptional target of the pluripotency factor OCT3/4, merase is an attractive target for cancer therapy (Kim et al., which binds the TRF1 promoter to up-regulate TRF1 expression, 1994; Shay, 2016). Different approaches have been designed in providing a mechanistic link between TRF1 and pluripotency the search for telomerase inhibitors: small-molecule inhibitors, (Schneider et al., 2013). Although TRF1 is clearly essential antisense oligonucleotides, G-quadruplex stabilizers, immu-

fuse. (D) Fused telomeres lead to a mitotic arrest checkpoint during which telomere dysfunction is amplified by Aurora B–dependent TRF2 removal, causing cell death through apoptosis, necrosis, or autophagy in crisis, a second proliferative barrier that protects against tumor development. (E) Reactivation of either telomerase activity or ALT in some very rare crisis cells allows these premalignant cells to escape crisis and divide unlimitedly (immortalization). Fused chromosomes in postcrisis cells lead to large-scale genomic rearrangements that promote acquisition of oncogenic mutations and malignant traits required for a fully malignant phenotype, cancer.

882 JCB • Volume 216 • Number 4 • 2017 notherapy, gene therapy using telomerase promoter-driven ex- most of the cancer cell lines assayed and to telomere dysfunc- pression of a suicide gene, and chemicals that block telomerase tion in in vivo xenograft models (Mender et al., 2015). Target- biogenesis (Harley, 2008; Agrawal et al., 2012; Buseman et al., ing telomerase recruitment to telomeres has also been proposed 2012; Jafri et al., 2016). Among all antitelomerase compounds as a potential anticancer treatment (Nakanishi et al., 2003). developed, the oligonucleotide imetelstat (GRN163L) appears We have recently developed a novel telomerase-inde- to be the most promising telomerase inhibitor and the one most pendent and telomere length–independent strategy to induce extensively evaluated in clinical trials. Imetelstat sequence telomere dysfunction by targeting TRF1 (Fig. 2; García-Bec- binds to a complementary oligonucleotide region of hTR at the caria et al., 2015). Genetic Trf1 deletion impaired the growth of active site of telomerase holoenzyme, leading to complete inhi- p53-null KRas(G12V)-induced lung carcinomas and increased bition of enzyme activity (Fig. 2; Jafri et al., 2016). However, mouse survival independently of telomere length. Chemical inhibition of telomere maintenance by targeting telomerase in inhibition of TRF1 binding to telomeres by small molecules cancer has showed effectiveness in only some myeloid tumors blocked the growth of already established lung carcinomas by but has largely failed in solid tumors (Baerlocher et al., 2015; inducing a rapid DDR and without affecting mouse survival Tefferi et al., 2015; Jafri et al., 2016). The reasons underlying or tissue function, supporting induction of acute telomere un- the lack of success are likely that therapeutic strategies based capping as a promising therapeutic target for lung cancer and on telomerase inhibition to treat cancer will be effective only very likely for many other cancer types (García-Beccaria et al., when telomeres shorten below a minimum length, implying a 2015). Chemical inhibition of TRF2 dimerization has also been long lag period before cell death. Indeed, telomerase activity achieved by targeted peptide synthesis that directly binds to the is dispensable for transformation of cells with long telomeres TRFH dimerization domain of TRF2 (Di Maro et al., 2014). (Seger et al., 2002), and studies with telomerase inhibitors indi- Treatment of HeLa cells with TRF2-binding chemotypes did cate that they are effective preferentially in cells with short telo- not affect TRF2 telomeric localization but induced rapid DDR meres (Wang et al., 2004; Buseman et al., 2012). In line with activation, end-to-end fusions, and cell death indicative of this idea, telomerase abrogation in the context of cancer-prone telomere dysfunction (Di Maro et al., 2014). Although further mouse models, including the K-Ras+/G12D lung tumorigene- clinical research is needed to ultimately address the efficacy sis mouse model, showed antitumorigenic activity only after of telomere-uncapping chemical inhibition in human cancer, several mouse generations in the absence of telomerase when these strategies provide novel opportunities for the develop- telomeres reached a critically short length (Chin et al., 1999; ment of anticancer agents. Greenberg et al., 1999; González-Suárez et al., 2000; Perera et al., 2008). Moreover, these antitumorigenic effects of short Outstanding questions in telomere-related telomeres owing to telomerase deficiency are abrogated in the diseases and cancer absence of p53 (Chin et al., 1999). Although telomere biology has been extensively studied, our Alternative therapeutic approaches that target telomeres in knowledge of the implications of telomeres and telomere pro- a telomere length–independent manner are starting to emerge. teins in human disease has considerably improved in recent In contrast to telomerase inhibition, telomere uncapping has years. Current research efforts focus on how our knowledge of been shown to cause rapid induction of cell death and/or senes- telomere biology and its connection with human disease can be cence in a manner that is independent of telomerase activity and translated into the clinic to improve human health. Although telomere length (Karlseder et al., 1999; Martínez et al., 2009b). some telomere-based therapies have already reached clinical Thus, given that telomere dysfunction can be achieved inde- trials, the path is still long. The fact that telomere dysfunc- pendently of telomere length, telomere uncapping strategies tion may have different and even opposing outcomes in aging emerge as a more universal way to rapidly impair the growth of and cancer makes the goal of developing telomere-based anti- dividing cells. One of these alternative approaches consists of aging and anticancer therapies a delicate challenge that re- a telomere-targeted telomerase-dependent potential anticancer quires a highly controllable technology. Results obtained in therapy that uses a nucleoside analogue, 6-thio-2′-deoxygua- mouse models with telomerase gene therapy as a treatment for nosine (6-thio-dG). 6-Thio-dG is recognized by telomerase aging-associated diseases and telomere syndromes, as well as and incorporated into de novo synthesized telomeres, leading shelterin chemical inhibition as anticancer therapy treatment, to telomere dysfunction solely in telomerase-expressing cells constitute promising opportunities for further clinical develop- (Fig. 2). Treatment with 6-thio-dG leads to rapid cell death in ment. However, additional studies are needed to determine the

Table 2. Examples of mouse models underscoring telomere dysfunction as either a driving force in cancer development or as a cancer suppressor

Genotype Cancer phenotype Reference

Terc−/− p53−/− Earlier onset lymphoma/sarcoma, epithelial carcinoma Chin et al., 1999 Terc−/− Msh2−/− Lymphoma/carcinoma Martinez et al., 2009a Trf1lox/lox K5-cre p53−/− Squamous cell carcinoma Martínez et al., 2009b Pot1alox/lox p53lox/lox hCD2-iCre T cell lymphomas Pinzaru et al., 2016 Pot1alox/lox p53lox/lox Sprr2f-Cre Endometrial adenocarcinomas Akbay et al., 2013 Tpp1Acd p53−/− Carcinomas Else et al., 2009 Tpp1lox/lox K5-cre p53−/− Cancer suppressor Tejera et al., 2010 Terc−/− Ink4a/Arf−/− Delayed lymphoma/fibrosarcoma onset Khoo et al., 2007 Trf1lox/lox p53−/− K-RasLSLG12V Growth impairment of lung carcinoma García-Beccaria et al., 2015

The effects of telomere dysfunction in cancer-prone mouse models depend on the status of DNA damage surveillance mechanisms, acting oncogenes, and tissue-specific factors.

Telomeres as therapeutic targets • Martínez and Blasco 883 undesired long-term effects associated with exogenous reex- Alter, B.P., P.S. Rosenberg, N. Giri, G.M. Baerlocher, P.M. Lansdorp, and S.A. Savage. 2012. Telomere length is associated with disease severity pression of telomerase and shelterin inhibition. and declines with age in dyskeratosis congenita. Haematologica. 97:353– The molecular mechanisms underlying the specificity of 359. http​://dx​.doi​.org​/10​.3324​/haematol​.2011​.055269 certain specific disease-associated mutations/variants in shelter- Aoude, L.G., A.L. Pritchard, C.D. Robles-Espinoza, K. Wadt, M. Harland, ins are yet an open field of research. For example, specific POT1 J. Choi, M. Gartside, V. Quesada, P. Johansson, J.M. Palmer, et al. 2014. Nonsense mutations in the shelterin complex genes ACD and TERF2IP variants give rise to chronic lymphocytic leukemia, whereas in familial melanoma. J. Natl. Cancer Inst. 107:dju408. http​://dx​.doi​.org​ other amino acid substitutions predispose to familial melanoma, /10​.1093​/jnci​/dju408 cardiac angiosarcomas, or gliomas. Several shelterin mutations Armanios, M., and E.H. Blackburn. 2012. The telomere syndromes. Nat. Rev. associated with different tumor types have recently been found, Genet. 13:693–704. http​://dx​.doi​.org​/10​.1038​/nrg3246 and presumably many more shelterin mutations are yet to be Armanios, M.Y., J.J. Chen, J.D. Cogan, J.K. Alder, R.G. Ingersoll, C. Markin, W.E. Lawson, M. Xie, I. Vulto, J.A. Phillips III, et al. 2007. Telomerase identified in human cancer. These observations further support mutations in families with idiopathic pulmonary fibrosis. N. Engl. J. Med. the use of shelterins as cancer targets and raise the question of 356:1317–1326. http​://dx​.doi​.org​/10​.1056​/NEJMoa066157 the potential translational applications of shelterin gene therapy Artandi, S.E., and R.A. DePinho. 2010. Telomeres and telomerase in cancer. as preventive treatment in mutation carriers. Carcinogenesis. 31:9–18. http​://dx​.doi​.org​/10​.1093​/carcin​/bgp268 In summary, the works discussed in this review provide Baerlocher, G.M., E. Oppliger Leibundgut, O.G. Ottmann, G. Spitzer, O. Odenike, M.A. McDevitt, A. Röth, M. Daskalakis, B. Burington, proof of concept for the feasibility of telomere-targeted thera- M. Stuart, and D.S. Snyder. 2015. Telomerase inhibitor imetelstat in peutic approaches for the prevention and treatment of telomere patients with essential thrombocythemia. N. Engl. J. Med. 373:920–928. dysfunction–mediated diseases in animal models and pave the http​://dx​.doi​.org​/10​.1056​/NEJMoa1503479 way for their development and implementation in the clinic. Bainbridge, M.N., G.N. Armstrong, M.M. Gramatges, A.A. Bertuch, S.N. Jhangiani, H. Doddapaneni, L. Lewis, J. Tombrello, S. Tsavachidis, Y. Liu, et al. Gliogene Consortium. 2014. 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